Lecture 5 - Nucleic Acid Detection Quantification and Titration Flashcards
How is PCR used in molecular diagnostics for viral detection?
Principle:
* Detects nucleic acids (DNA/RNA) in a sample.
* Requires knowledge of the target organism’s gene sequence.
Applications:
* Latent infections – Detects dormant virus DNA/RNA.
* Low quantities – Can detect very small amounts of DNA/RNA.
Limitations:
* PCR only detects what it’s designed to find – must already know the specific target.
What is viral quantification (titer), and why is it important in diagnostics?
Definition:
* Viral load (or viral burden) is the quantity of virus present in a given volume.
* Expressed as viral particles, such as plaque-forming units (PFU) or infectious particles per mL (TCID50), depending on the assay.
Importance:
* Used in vaccine development to determine the amount of virus required for effective immune response.
* Helps measure seroconversion – detecting the appearance of antibodies in response to infection or vaccination.
Antibody Titers:
* Titers express the reverse dilution at which antibodies can still be detected.
* For example, a 1:40 dilution where antibodies are still detectable means the titer = 40.
What is PCR, and how is it used in diagnostics?
Purpose:
* Amplifies specific DNA/RNA sequences for detection or analysis.
* Widely used in molecular diagnostics to detect pathogens.
Steps in PCR:
* Denaturation – DNA is heated to separate strands.
* Annealing – Primers bind to specific target sequences.
* Extension – DNA polymerase synthesizes new DNA strands.
Applications:
* Detects specific genes (e.g., viral genomes) in samples.
* Can identify latent infections or small quantities of DNA/RNA.
Limitation:
* Target-specific – only detects the sequence you know; must design primers for the exact sequence of interest.
What is sequencing, and how is it used in viral diagnostics?
Principle:
* Automated process used to determine the base pair sequence of nucleic acids (DNA/RNA).
* PCR products are often used as templates for sequencing.
Applications:
* Genomic sequencing of viruses and bacteria to understand their genetic makeup.
* Metagenomics – sequences all nucleic acids in a sample (host and pathogen) to identify unknown pathogens (e.g., Schmallenberg virus).
Use in Diagnostics:
* Detection of new/unknown viruses by identifying sequences not yet known or cataloged.
What is Real-Time PCR, and how is it used in molecular diagnostics?
Principle:
* Measures the production of PCR products as the reaction progresses.
* Fluorescent signal is emitted, proportional to the amount of PCR product produced.
Applications:
* Allows quantification of DNA/RNA by measuring fluorescence intensity.
* Quantitative assay – measures gene expression levels or pathogen load.
Common Assays:
* SybrGreen assay – binds to double-stranded DNA, emitting fluorescence.
* TaqMan assay – uses a fluorescent probe that binds specifically to the target DNA sequence.
Advantages:
* Provides real-time data for monitoring the reaction.
* Allows for quantitative analysis of the target DNA or RNA.
What is a Plaque Forming Assay (PFU), and how is it used in viral diagnostics?
Purpose:
* Measures the number of infectious viruses in a given volume of a sample.
* Assesses the ability of the virus to form plaques (zones of cell lysis) on a monolayer of susceptible cells.
How it Works:
* A diluted virus is applied to a cell culture.
* The virus infects and lyses cells, forming distinct plaques.
* The number of plaques corresponds to the number of infectious viral particles (PFU).
Expression:
* Results are expressed as the number of viruses per volume (e.g., PFU/mL).
What is virus titration, and how is it performed?
Purpose:
* Determines the concentration of infectious virus particles in a sample.
* Helps assess viral load and virus infectivity.
Methods:
* Plaque Forming Assay (PFU): Counts the number of plaques formed by virus infection in a cell culture.
* TCID50 (Tissue Culture Infective Dose): Measures the dilution at which 50% of cultured cells are infected.
Steps:
* Dilute the virus sample in a series of concentrations.
* Infect cell cultures with the diluted samples.
* Count plaques (PFU) or observe the cytopathic effect (CPE) to estimate viral concentration.
Importance:
* Provides accurate estimates of viral infectivity.
* Used in vaccine development and virus production for research.
What is TCID50, and how is it used to measure the infectious nature of a virus?
Definition:
* A statistical value that quantifies the infectious nature of a virus.
* Represents the dilution of a virus sample that causes cytopathic effects (CPE) in 50% of inoculated cells or kills 50% of infected hosts.
Measurement:
* The virus suspension is diluted to determine the endpoint where 50% of tissue culture cells exhibit CPE or are infected.
* TCID50 is the dilution level where 50% of the infected cultures show signs of infection.
Calculation Methods:
* Spearman-Karber method or Reed and Muench method are commonly used formulas to calculate TCID50 values.
Importance:
* Provides a quantitative measure of viral infectivity.
* Commonly used in virology to assess viral load and potency for research or vaccine development.
